Cancer is currently the second leading cause of death in the United States. Metastatic cancers are especially difficult to treat and are associated with higher levels of mortality compared to benign tumors. American men and women have a 38-44% chance of developing invasive cancers over the course of their lifetimes. Tumor metastasis involves cancer cell invasion of surrounding tissues, often accelerated by cell surface proteases. One such protease known as the urokinase-type plasminogen activator (uPA) is capable of breaking down extracellular matrix proteins and activating migration-inducing signal cascades through binding to the urokinase-type plasminogen activator receptor (uPAR).
A large body of evidence suggests that uPA and uPAR expression are substantially higher on invasive malignant cancer cells than on healthy cells or benign tumors. In clinical settings, high levels of uPAR are used as diagnostic measures for metastatic potential and poor clinical outcome in several malignancies. Novel strategies to combat cancer are highly desirable due to the limitations of the more traditional treatment options, including radiation therapy and chemotherapy. These treatment methods are not only associated with significant side effects but are also limited with respect to their effectiveness in the treatment of late stage cancers.
The ability to target cancer cells selectively is thus of great importance, with the potential to significantly reduce toxicity and off-target effects, thereby reducing side effects experienced by the patient. New approaches to treat cancer that combine the advantages of traditional small molecules and biologics could address many of the limitations associated with currently available therapies.
Anthroquinone-based small molecules have been identified previously as potential anti-cancer agents and have been shown to bind to uPAR in vitro with triple digit nM affinity capable of blocking cancer cell invasion, migration, and adhesion at double to triple uM concentrations (T. Mani et al). Previously, the development of an antineoplastic antibody-recruiting agent equipped with the urokinase protein as a uPAR target-binding domain was reported from the present laboratory. The molecule developed here, termed “ARM-U,” was shown previously to selectively target uPAR-over-expressing A172 glioblastoma cells and facilitate anti-DNP antibody-dependent cellular phagocytosis (ADCP) at single digit nM concentrations. Although ARM-U has high-affinity binding to uPAR (Kd of ˜200 pM), its potential as a therapeutic is limited due to the incorporation of the large uPA protein with limited stability in vivo. This, plus the limitations associated with the administration of uPA protein limits its use.
The present invention describes the rational design of the highest affinity small molecule targeting the uPA site on uPAR ever reported. Novel small molecule derived ARM-U2 compounds demonstrate efficacy against metastatic cancer cells in cellular assays at low nM concentrations. Moreover, these compounds do not require the administration of uPA protein in order to facilitate its therapeutic efficacy, a clear advance over the prior art compounds.
The present invention also illuminates the nature of the non-covalent interactions with the uPA binding site on uPAR required for tight binding to uPAR. In addition, the present invention has provided experimentally supported computational predictions regarding the importance of basic residues in the uPA binding pocket for targeting small molecules selectively to uPAR and reports the first crystal structure of a uPAR targeting antibody recruiting small molecule binding the uPA binding site of uPAR. An additional feature of the present invention is that it demonstrate the high potency and efficacy of an antibody recruiting small molecule.
The present invention shows that (ARM-U2) binds uPAR on the surface of A172 glioblastoma and B16 melanoma cancer cells and recruits endogenous anti-DNP antibodies to the cell surface which in turn induce antibody dependent cellular phagocytosis and antibody dependent cellular cytotoxicity (see FIG. 1). Present preliminary in-vivo data show the ability of ARM-U2 to inhibit tumor formation using a B-16 xenograft mouse tumor model